Prekallikrein is a single-chain glycoprotein which is present in human blood in at least two forms having molecular weights of 85 kDa and 88 kDa, respectively. Prekallikrein is one of five plasma proteins which are junction-initiating and/or modulatory proteins for coagulation, fibrinolysis, complement activation, prorenin activation, and possibly other biochemical pathways occurring in the plasma. Colman, R. W., J. Clin, Inv. 73:1249-1253 (1984). The other major junction-initiating or modulatory plasma proteins are (1) factor XII, also known as Hageman factor, (2) high molecular weight kininogen, (3) Cl-inhibitor and (4) alpha.sub.2 -macroglobulin.
Prekallikrein and factor XII are plasma serine protease zymogens. Conversion of prekallikrein to the active enzyme kallikrein is initiated by factor XII autoactivated to factor XIIa in the presence of negatively charged surfaces. Factor XIIa or an activated fragment of factor XII (factor XIIf) cleaves prekallikrein into kallikrein. Kallikrein activates factor XII to produce additional factor XIIa. Kallikrein consists of two polypeptides linked together by one or more disulfide bridges. One of the kallikrein polypeptides, known as the "heavy chain", has a molecular weight of 53 kDa. The other polypeptide, known as the "light chain" has two variants, 33 kDa and 37 kDa, respectively, depending on the form of prekallikrein from which they originate. The light chain contains the enzyme active site. The 33 kDa kallikrein light chain originates from the 85 kDa prekallikrein, while the 37 kDa variant is from the 88 kDa form of prekallikrein.
Prekallikrein is present in blood as a non-covalent complex with high molecular weight kininogen. The latter is a cofactor for both factor XIIa and kallikrein. It is also a substrate of kallikrein. Kallikrein cleaves high molecular weight kininogen into four fragments: three polypeptides and the potent vasodepressor peptide bradykinin. Other substrates of kallikrein include plasminogen and prorenin. In addition, kallikrein stimulates neutrophil aggregation and degranulation.
Cl-inhibitor and alpha.sub.2 -macroglobulin are the two major plasma protease inhibitors. Other inhibitors such as antithrombin III can contribute about 13% to kallikrein inhibition of surface-activated normal plasma in vitro. Inhibition by these minor inhibitors may be higher in situations where Cl-inhibitor and alpha.sub.2 -macroglobulin are depleted or inactivated.
Cl-inhibitor inhibits kallikrein by forming a covalent complex with it. Alpha.sub.2 -macroglobulin inhibits kallikrein by combining with kallikrein to form a partially inactive complex. Cl-inhibitor and alpha.sub.2 -macroglobulin thus regulate the activity of kallikrein in the body. Kallikrein is also inhibited by combining with antithrombin III.
A deficiency of Cl-inhibitor gives rise to the classic disease hereditary angioedema and a decrease in the level of functional prekallikrein without change in prekallikrein/kallikrein antigen. Activation of prekallikrein may be inferred from a drop in its plasma level, by formation of kallikrein-Cl-inhibitor complex, or by the release of bradykinin into the circulation, concomitant with cleavage of plasma high molecular weight kininogen. The appearance of these signals has been demonstrated in a variety of diseases, most notably gram negative septicemia. Colman, R. W., J. Clin. Inv. 73:1249-1253 (1984). This affliction is a major cause of death and disability among hospitalized patients, with an incidence of several hundred thousand cases each year. Hypotension and hemorrhage are frequent complications due to activation of the contact phase of blood coagulation. Endotoxin infusion leads to activation of factor XII, which in turn cleaves prekallikrein to the active form kallikrein, resulting in a drop in the level of prekallikrein in the plasma. When kallikrein inhibitors are consumed, the concentration of the active form, kallikrein (as distinguished from the inactive form, prekallikrein), is increased. Early detection of this change in plasma prekallikrein and kallikrein levels allows intervention with treatment before the occurrence of irreversible shock.
Kohler and Milstein, Nature 256, 493-497 (1975) were the first to describe the fusion of myeloma cells to immune spleen cells from mice to generate continuous cell lines. These hybrid cell lines, or hybridomas, have characteristics that neither the parental myeloma cells nor parental immune spleen cells possess. Hybridomas are capable of continuously producing homogeneous (monoclonal) antibodies which recognize a single antigenic determinant. Prior to the work of Kohler and Milstein, only polyclonal antisera could be obtained, which are not capable of continuously producing identical antibodies. Polyclonal antibodies recognize a number of different antigenic determinants. Because of homology among the polypeptides containing the active site of several serine proteases, and homology between the heavy chains of prekallikrein and factor XI, thorough characterization of antibodies is necessary for diagnostic utility, which could be difficult and time-consuming in the case of polyclonal antibodies.
Although techniques for the production of hybridomas are now extensively described in the literature, e.g., Monoclonal Antibodies, Hybridomas: A New Dimension In Biological Analysis, R. H. Kennett, T. J. McKearn, and K. B. Bechtol, eds., Plenum Press, New York and London (1980), there is no general method for obtaining successful monoclonal antibody-producing hybridomas which can be used with all antigens. Fusion techniques must be varied in each case to obtain hybridomas producing monoclonal antibody to the desired antigen. In order to obtain antibodies specific to a single antigen, laborious purification techniques are required to provide highly purified antigen for either immunization or screening. The production of monoclonal antibodies for any given antigen is still a highly empirical process.
There have been no previous reports of monoclonal antibodies against human plasma prekallikrein, although plasma prekallikrein is present in all humans with very few exceptions. Prekallikrein and kallikrein are detected in patient blood by means of coagulant and immunochemical assays using polyclonal antisera. The dearth of literature accounts of monoclonal antibodies to prekallikrein is no doubt due to difficulties in the purification of antigen and/or the lack of success in preparation of suitable hybridomas.